Spectramax m2e

For cellular ROS detection, cells were incubated with 2,7 dichloro-fluorescein diacetate (DCFDA) (Abcam) 100 μM for 30 min at 37 °C in the dark, and fluorescence was analyzed with excitation/emission at 495/529 nm, using SpectraMax M2e (Molecular devices). Fluorescence intensity was then normalized for cell number. To determine mitochondrial ROS production, cells were treated with 5 μM MitoSOX^TM Red, a mitochondrial superoxide indicator (Invitrogen) for 10 min at 37 °C, according to the manufacturer’s protocol, and fluorescence was analyzed with excitation/emission at 510/580 nm, using SpectraMax M2e (Molecular devices).

Embryos or newly hatched L1 larva (which were less than 2hrs post hatching) were treated in 50% bleach for two minutes, then collected in 50μL of TSS (0.4%w/v NaCl, 0.03%v/v Triton X-100). 10 individuals were collected per replicate. The BODIPY C11 (Thermo) solution was made by adding 1μL of 10μg BODIPY C11/μL DMSO to 2mL of Grace’s Insect Medium (Sigma-Aldrich). 100 μL of Grace’s Medium + C11 were then added to each replicate. These samples were homogenized for 20s using a motorized pestle. Samples were briefly spun down then allowed to incubate at 21°C in the dark for 30min. Samples were then measured on a Molecular Devices’ SpectraMax M2e, set to capture two fluorescent readings: red excitation 561nm/ auto cutoff 590/ emission 591 and green excitation 485nm/ auto cutoff 495/ read 510nm. Data is represented as the green reading divided by the red reading.
TMRE assays were performed with the above protocol with 1μL of 10μM TMRE (Biotium) in DMSO replacing the BODIPY C11. Samples were imaged without and incubation step on a Molecular Devices’ SpectraMax M2e set to capture one reading at excitation 549nm/ cutoff 570nm/ emission 575nm.

For cellular ROS detection, cells were incubated with 2,7 dichloro‐fluorescin diacetate (DCFDA) (Abcam) 100 μM for 30 min at 37°C in the dark, and fluorescence was analyzed with excitation/emission at 495/529 nm, using SpectraMax M2e (Molecular devices). Fluorescence intensity was then normalized for cell number. To determine mitochondrial ROS production, cells were treated with 5 μM MitoSOX™ Red, a mitochondrial superoxide indicator (Invitrogen) for 10 min at 37°C, according to the manufacturer's protocol, and fluorescence was analyzed with excitation/emission at 510/580 nm, using SpectraMax M2e (Molecular devices).

Interleukin (IL)-4, IL-10, and interferon (IFN)-γ in the serum of immunized mice were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). Standard curves were generated for each cytokine. Values were measured at an optical density of 450 nm using an ELISA plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA). Antigen-specific immunoglobulin (Ig)G from serum or IgA from fecal samples was measured using ELISA based on our previous study (Hwang et al., 2023 (link)). Briefly, each well of a 96-well plate was pre-coated with E. coli-expressed recombinant SARS-CoV-2 spike S1 antigens (1 µg per well) overnight at 4°C. The antigen-coated wells were blocked with bovine serum albumin for 1 h at 37°C. After blocking, immunized mouse serum (1:100 dilution) or fecal extract (1:10 dilution) was added to the wells and incubated at 37°C for 1 h. HRP-conjugated goat anti-mouse IgG or IgA antibody (1:1000; Invitrogen, Waltham, MA, USA) was added to the wells and incubated for 1 h at 37°C. The antigen-specific antibody was detected by adding 3, 3’, 5, 5’- tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA). The reaction was stopped by adding 0.5 N H₂SO₄. Optical density (450 nm) was measured using an ELISA plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA, USA).

For cellular ROS detection, cells were incubated with 2,7 dichloro- fluorescein diacetate (DCFDA) (Abcam) 100 μM for 30 min at 37 °C in the dark, and fluorescence was analyzed with excitation/emission at 495/529 nm, using SpectraMax M2e (Molecular devices). Fluorescence intensity was then normalized for cell number. To determine mitochondrial ROS production, cells were treated with 5 μM MitoSOX™ Red, a mitochondrial superoxide indicator (Invitrogen) for 10 min at 37 °C, according to the manufacturer’s protocol, and fluorescence was analyzed with excitation/emission at 510/580 nm, using SpectraMax M2e (Molecular devices).